Vacuolar membrane localization of the Arabidopsis‘two‐pore’ K⁺ channel KCO1

Abstract

SummaryPotassium (K + ) channels play multiple roles in higher plants, and have been characterized electrophysiologically in various subcellular membranes. The K + channel AtKCO1 from Arabidopsis thaliana is the prototype of a new family of plant K + channels. In a previous study the protein has been functionally characterized after heterologous expression in Baculovirus-infected insect cells. In order to obtain further information on the physiological function of AtKCO1, the gene expression pattern and subcellular localization of the protein in plants were investigated. The regulatory function of the 5¢ region of the AtKCO1 gene was examined in transgenic A. thaliana plants carrying b-glucuronidase (GUS) fusion constructs. Our analysis demonstrates that the AtKCO1 promoter is active in various tissues and cell types, and the highest GUS activity could be detected in mitotically active tissues of the plant. Promoter activity was strongly dependent on the presence of a 5¢ leader intron. The same overall structure was identi®ed in two genes encoding AtKCO1-like K + channels from Solanum tuberosum (StKCO1a and StKCO1b). To investigate the subcellular localization of AtKCO1, the channel protein, as well as a fusion protein of AtKCO1 with green¯uorescence protein (GFP), were expressed in transgenic tobacco BY2 cells. In sucrose density gradients, both proteins co-fractionate with tonoplast markers (NtTIPa, vATPase). In¯uorescence images from transgenic AtKCO1±GFP BY2 cells¯uorescence was exclusively detected in the tonoplast. Thus AtKCO1 is the ®rst cloned K + channel demonstrated to be a vacuolar K + channel.

TheArabidopsisMATE Transporter TT12 Acts as a Vacuolar Flavonoid/H+-Antiporter Active in Proanthocyanidin-Accumulating Cells of the Seed Coat

“…While expression of the unmodified TT12 cDNA fully complemented the tt12 mutant phenotype (including returning Q3R and insoluble and soluble PAs to wild-type levels; Figure 2), the Pro35S-dual:GFP5:TT12:t35S construct restored flavonoid levels at ;75, 37, and 20% of the wild-type amount of Q3R and insoluble and soluble PAs, respectively (Figure 2), suggesting that a significant portion of GFP-cTT12 protein reached its proper destination within the cell and was functional. Transient expression by biolistic particle delivery of GFPcTT12 together with the established tonoplast marker TPK1 (Czempinski et al, 2002) fused to DsRed2 in onion bulb epidermis cells demonstrated that both proteins coreside on membranes (see Supplemental Figure 1A online). Importantly, fluorescence of both fusion proteins is visible on strands traversing the vacuole.…”

Roles of ion channels and transporters in guard cell signal transduction

“…VK currents may be mediated by members of the TPK/KCO K + channel family, encoded in Arabidopsis by six genes. KCO1/TPK1 was the first K + channel to be localized to the plant tonoplast [145] and, like VK currents of guard cells, when heterologously expressed in yeast KCO1 channels exhibit K + selectivity, activation by Ca 2+ , and voltage independence [146].…”

Novel Tonoplast Transporters Identified Using a Proteomic Approach with Vacuoles Isolated from Cauliflower Buds

“…4). As a vacuolar control we used the C-terminal DsRed-tagged KCO1 channel from Arabidopsis (At5g55630), known to reside in the vacuolar membrane (Czempinski et al, 2002).…”